Project Summary It is well-accepted that deregulated immunity is a major contributor to tumor growth and progression. Tumor cells have been demonstrated to transform the microenvironment into an immune-suppressive niche to facilitate their growth. Therefore, dissecting the mechanisms by which tumor cells regulate the immune compartment is instrumental to providing insights for the improvement and development of immune therapies. In the thesis work of this project, I am investigating the roles of Eya3 in immunosuppression and tumor progression. Eya3 is a member of a family of multifunctional Eya proteins, which contain transactivating potential, and completely separable Tyrosine (Tyr) and Threonine (Thr) phosphatase activities. Interestingly, their expression is not only associated with normal development, but is often altered in tumors. Eya3 is most expressed in the highly aggressive form of breast cancer, triple negative breast cancer (TNBC). The role of the Thr phosphatase activity of Eya in cancer has not been studied, yet its transactivating potential and Tyr phosphatase activity have both been implicated in breast cancer metastasis. My studies on the Thr phosphatase activity of Eya3 have resulted in novel findings. First, I have shown that in contrast to the previous notion that Eya3 possesses intrinsic Thr phosphatase activity, Eya3 interacts with PP2A to exhibit Thr phosphatase activity. This associated activity enhances c-Myc stability, resulting in upregulation of the immune checkpoint protein PD-L1, dampening anti-tumor T-cell responses. This work is the first demonstration of a role for TNBC expressed Eya3 in regulating adaptive immunity and tumor progression. In this F99/K00 proposal, I describe a research strategy to complete my dissertation studies, and outline the direction I would like to take in my postdoctoral years. In Aim 1a, I describe my findings to date, which are described above and have resulted in one co-first author publication in Nature Communications (in press) as well as a second author publication in revision at J Clin Invest. Aim 1b outlines my research plan for completion of my dissertation studies, where I examine which activity of Eya3 is most required for its tumor promotional effects (with the idea that this will aid in therapeutic targeting of the correct activity), and I will further identify which targets (using both a targeted and unbiased approach) of Eya3 mediate phenotypes associated with various activities. In Aim 2, I focus on what I would like to work on in my postdoctoral years (K00). It has been reported that tumor cells can disseminate from a pre-malignant lesion, and these disseminated tumor cells (DTC) are able to remain dormant, and eventually re-grow into lethal metastases. Thus, gaining a better understanding of the mechanisms by which tumor cells maintain and exit dormancy will provide insights for developing therapeutic strategies to keep tumor cells in dormancy or to specifically eliminate dormant tumor cells. Aim 2 focuses on exploring how the dynamic tumor microenvironment impinges on dormancy of DTCs, in an attempt to identify druggable molecules, unique to tumor-stroma crosstalk, that are necessary for the break of dormancy.